mouse brain cdna matchmaker lexa library  (TaKaRa)

Journal: Journal of Biological Chemistry

Article Title: The Chaperone Protein 14-3-3η Interacts with the Nicotinic Acetylcholine Receptor α4 Subunit

doi: 10.1074/jbc.m011549200

Figure Lengend Snippet: FIG. 4. 14-3-3h stabilizes the a4 wild-type subunit and not the mutant a4S441A subunit. Cells were cotransfected with the a4 cDNA (1st to 4th lanes) or a4S441A cDNA (5th to 8th lanes), with 14-3-3h cDNA (2nd, 4th, 6th, and 8th lanes) or without 14-3-3h cDNA (vector pEF6A alone) (1st, 3rd, 5th, and 7th lanes). The following day, the cells were treated with or without forskolin (Forsk, 10 mM). The bar across the paired lanes represents samples treated under identical conditions from independently transfected and processed samples from a single experiment. Forty eight hours after transfection, the cells were lysed in 500 ml of 2% Nonidet P-40 lysis buffer, and the proteins were solubilized for 3 h. The lysates were centrifuged, and 20 ml of supernatant were fractionated by SDS-PAGE. The top half of the blots was immuno- blotted (IB) with the anti-a4 subunit mAb and the bottom half with the anti-14-3-3 mAb.

Article Snippet: Approximately 2 3 106 yeast cells cotransformed with the bait and cDNAs from a premade mouse brain cDNA Matchmaker LexA library (CLONTECH Laboratories Inc., Palo Alto, CA) were screened.

Techniques: Mutagenesis, Plasmid Preparation, Transfection, Lysis, SDS Page